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Results for “Subcellular localization”

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PubMed2002

Characterization of subcellular localization and stability of a splice variant of G alpha i2.

BACKGROUND: Alternative mRNA splicing of alpha(i2), a heterotrimeric G protein alpha subunit, has been shown to produce an additional protein, termed salpha(i2). In the salpha(i2) splice variant, 35 novel amino acids replace the normal C-terminal 24 amino acids of alpha(i2). Whereas alpha(i2) is found predominantly at cellular plasma membranes, salpha(i2) has been localized to intracellular Golgi membranes, and the unique 35 amino acids of salpha(i2) have been suggested to constitute a specific targeting signal. RESULTS: This paper proposes and examines an alternative hypothesis: disruption of the normal C-terminus of alpha(i2) produces an unstable protein that fails to localize to plasma membranes. salpha(i2) is poorly expressed upon transfection of cultured cells; however, radiolabeling indicated that alpha(i2) and salpha(i2) undergo myristoylation, a co-translational modification, equally well suggesting that protein stability rather than translation is affected. Indeed, pulse-chase analysis indicates that salpha(i2) is more rapidly degraded compared to alpha(i2). Co-expression of betagamma rescues PM localization and increases expression of salpha(i2). In addition, alpha(i2)A327S, a mutant previously shown to be unstable and defective in guanine-nucleotide binding, and alpha(i2)(1-331), in which the C-terminal 24 amino acids of alpha(i2) are deleted, show a similar pattern of subcellular localization as salpha(i2) (i.e., intracellular membranes rather than plasma membranes). Finally, salpha(i2) displays a propensity to localize to potential aggresome-like structures. CONCLUSIONS: Thus, instead of the novel C-terminus of salpha(i2) functioning as a specific Golgi targeting signal, the results presented here indicate that the disruption of the normal C-terminus of alpha(i2) causes mislocalization and rapid degradation of salpha(i2).

Alternative Splicing↗

PubMed1993

Subcellular localization of Cdc42p, a Saccharomyces cerevisiae GTP-binding protein involved in the control of cell polarity.

The Saccharomyces cerevisiae Cdc42 protein, a member of the Ras superfamily of low-molecular-weight GTP-binding proteins, is involved in the control of cell polarity during the yeast cell cycle. This protein has a consensus sequence (CAAX) for geranylgeranyl modification and is likely to be associated, at least in part, with cell membranes. Using cell fractionation and immunolocalization techniques, we have investigated the subcellular localization of Cdc42p. Cdc42p was found in both soluble and particulate pools, and neither its abundance nor its distribution varied through the cell cycle. The particulate form of Cdc42p could be solubilized with detergents but not with NaCl or urea, suggesting that it is tightly associated with membranes. An increase in soluble Cdc42p was observed in a geranylgeranyltransferase mutant strain (cdc43-2ts) grown at the restrictive temperature. In addition, Cdc42p from a cdc42C188S mutant strain (that has an alteration at the prenylation consensus site) was almost exclusively in the soluble fraction, suggesting that membrane localization is dependent on geranylgeranyl modification at Cys-188. Immunofluorescence and immunoelectron microscopy experiments demonstrated that Cdc42p localizes to the plasma membrane in the vicinity of secretory vesicles that were found at the site of bud emergence, at the tips and sides of enlarging buds, and within mating projections (shmoo tips) in alpha-factor-arrested cells. These results indicate that Cdc42p is localized to the bud site early in the cell cycle and suggest that this localization is critical for the selection of the proper site for bud emergence and for polarized cell growth.

Amino Acid Sequence↗

PubMed1997

Prognostic significance of glutathione S-transferase pi expression and subcellular localization in human gliomas.

The glutathione S-transferase (GST)-pi gene is overexpressed in many human cancers and preneoplastic lesions and is associated with failure of cancer chemotherapy and poor patient survival. Although GST-pi overexpression in tumors of the central nervous system has been observed, the prognostic and/or clinical relevance of this overexpression has, to date, not been investigated. In this study, we analyzed the level of GST-pi expression and its subcellular localization in 61 primary gliomas and correlated the results with tumor histology, patient age, and patient survival. We observed a strong positive correlation between the level of GST-pi expression and tumor grade and between the presence of GST-pi in glioma cell nuclei and patient age. Univariate and multivariate Cox regression analyses and Kaplan-Meier curves showed the level of GST-pi expression and its nuclear localization to be inversely correlated with patient survival. Relative risk for death of patients with high versus low tumor GST-pi expression was 3.2 (P = 0.0069) by univariate analysis and 2.6 (P = 0.036) by multivariate analysis. The relative risk of death associated with the presence of nuclear GST-pi in glioma cells was 3.9 (P = 0.0001) by univariate analysis and 4.4 (P < 0.0001) by multivariate analysis. These data indicate that high GST-pi expression in tumor cells and the presence of the GST-pi protein in tumor cell nuclei are associated with clinically more aggressive gliomas and are strong predictors of poor patient survival.

Adolescent↗

PubMed1984

Subcellular localization and tissue distribution of sialic acid-forming enzymes. N-acetylneuraminate-9-phosphate synthase and N-acetylneuraminate 9-phosphatase.

The activities of N-acetylneuraminate 9-phosphate synthase and N-acetylneuraminate 9-phosphatase, the two enzymes involved in the final steps of the biosynthetic pathway of N-acetylneuraminic acid, were measured with the substrates N-acetyl[14C]mannosamine 6-phosphate and N-acetyl[14C]neuraminic acid 9-phosphate respectively. Subcellular localization studies in rat liver indicated that both enzymes are localized in the cytosolic fraction after homogenization in sucrose medium. To test the possibility of misinterpretation due to the hydrolysis of N-acetylneuraminic acid 9-phosphate by non-specific phosphatases, the hydrolysis of various phosphate esters by the cytosolic fraction was tested. Only p-nitrophenyl phosphate was hydrolysed; however, competition studies with N-acetylneuraminic acid 9-phosphate and p-nitrophenyl phosphate indicated that two different enzymes were involved and that no competition existed between the two substrates. In various other rat tissues N-acetylneuraminate-9-phosphate synthase and N-acetylneuraminate 9-phosphatase activities were detected, suggesting that N-acetylmannosamine 6-phosphate is a general precursor for N-acetylneuraminic acid biosynthesis in all the tissues studied.

Animals↗

PubMed1994

Expression, tissue distribution and subcellular localization of dehydrin TAS14 in salt-stressed tomato plants.

We previously isolated and characterized TAS14, and mRNA that is induced in tomato upon osmotic stress or abscisic acid (ABA) treatment and that shares expression and sequence characteristics with other dehydrin genes in different species. Affinity-purified antibodies against TAS14 protein were used to study the expression of TAS14 protein, both in seedlings and mature plants, its tissue distribution and its subcellular localization. TAS14 protein was not detected in 4-day-old seedlings but accumulated after ABA, NaCl or mannitol treatments. In NaCl-treated seedlings, some protein was detectable after 6 h of treatment and reached maximal levels between 24 and 48 h. Concentrations ranging from 5 to 12.5 g/l NaCl induced the protein to similar levels. In salt-stressed mature plants, TAS14 was expressed abundantly and continuously in aerial parts, but only slightly and transiently in roots. Immunocytochemical analysis of salt-treated plants showed TAS14 accumulated in adventitious root primordia and associated to the provascular and vascular tissues in stems and leaves. Immunogold electron microscopy localized TAS14 protein both in the cytosol and in the nucleus, associated to the nucleolus and euchromatin. Since TAS14 is a phosphoprotein in vivo, the classes of protein kinases potentially responsible for its in vivo phosphorylation were tested in in vitro phosphorylation assays. TAS14 protein was phosphorylated in vitro by both casein kinase II and cAMP-dependent protein kinase.

Abscisic Acid↗

PubMed2001

Subcellular localization of fragile X mental retardation protein with the I304N mutation in the RNA-binding domain in cultured hippocampal neurons.

1. Fragile X syndrome, the most common form of inherited mental retardation, is caused by the lack or dysfunction of fragile X mental retardation protein (FMRP). The 1304N mutation in the RNA-binding domain of FMRP results in an exceptionally severe form of mental retardation. 2. We have investigated the subcellular localization of FMRP and its 1304N-mutated form in cultured hippocampal neurons and PC12 cells, using immunofluorescence microscopy. In PC12 cells, FMRP was predominantly localized to the cytoplasm and also to the processes after differentiation by NGF. 3. In cultured hippocampal neurons, granular labeling was detected along the neuronal processes. 4. Double-labeling with synaptophysin antibody revealed FMRP at synaptic sites in neurons. 5. The 1304N mutation did not appear to affect the transport of FMRP to dendrites or its localization at synaptic sites. Thus, FMRP is a synaptic protein and the severe phenotype observed in the patient with the 1304N mutation is not produced by alterations in dendritic transport.

Animals↗

PubMed2003

Protein kinase C phosphorylates ribosomal protein S6 kinase betaII and regulates its subcellular localization.

The ribosomal protein S6 kinase (S6K) belongs to the AGC family of Ser/Thr kinases and is known to be involved in the regulation of protein synthesis and the G(1)/S transition of the cell cycle. There are two forms of S6K, termed S6Kalpha and S6Kbeta, which have cytoplasmic and nuclear splice variants. Nucleocytoplasmic shuttling has been recently proposed for S6Kalpha, based on the use of the nuclear export inhibitor, leptomycin B. However, the molecular mechanisms regulating subcellular localization of S6Ks in response to mitogenic stimuli remain to be elucidated. Here we present data on the in vitro and in vivo phosphorylation of S6Kbeta, but not S6Kalpha, by protein kinase C (PKC). The site of phosphorylation was identified as S486, which is located within the C-terminal nuclear localization signal. Mutational analysis and the use of phosphospecific antibodies provided evidence that PKC-mediated phosphorylation at S486 does not affect S6K activity but eliminates the function of its nuclear localization signal and causes retention of an activated form of the kinase in the cytoplasm. Taken together, this study uncovers a novel mechanism for the regulation of nucleocytoplasmic shuttling of S6KbetaII by PKC-mediated phosphorylation.

Active Transport, Cell Nucleus↗

PubMed1999

Human S100A11 exhibits differential steady-state RNA levels in various tissues and a distinct subcellular localization.

In order to analyze the steady-state RNA levels of S100A11 in different tissues, a cDNA fragment of human S100A11 was isolated from a cDNA library. The obtained fragment was labeled and hybridized to RNA isolated from various tissues. The Northern blot analysis revealed that S100A11 RNA levels varied from high in placenta, through intermediate in heart, lung, kidney, and most muscle samples, to barely detectable in brain. An efficient purification method for recombinant S100A11 yielding high quantities was developed. Furthermore, to examine the subcellular localization of this protein, the human polypeptide S100A11 antibodies were raised in rabbit. S100A11 was found to have a localization distinct from other S100 proteins examined, and is mostly localized in the nucleus, with slight variations among different glioblastoma cell types.

Adult↗

PubMed1999

The nuclear dot protein sp100, characterization of domains necessary for dimerization, subcellular localization, and modification by small ubiquitin-like modifiers.

The Sp100 and promyelocytic leukemia proteins (PML) are constituents of nuclear domains, known as nuclear dots (NDs) or PML bodies, and are both covalently modified by the small ubiquitin-related protein SUMO-1. NDs play a role in autoimmunity, virus infections, and in the etiology of acute promyelocytic leukemia. To date, little is known about the function of the Sp100 protein. Here we analyzed Sp100 domains that determine its subcellular localization, dimerization, and SUMOylation. A functional nuclear localization signal and an ND-targeting region that coincides with an Sp100 homodimerization domain were mapped. Sequences similar to the Sp100 homodimerization/ND-targeting region occur in several other proteins and constitute a novel protein motif, termed HSR domain. The lysine residue of the Sp100 protein, to which SUMO-1 is covalently linked, was mapped within and may therefore modulate the previously described HP1 protein-binding site. A consensus sequence for SUMOylation of proteins in general is suggested. SUMOylation strictly depended on a functional nuclear localization signal but was not necessary for nuclear import or ND targeting. A three-dimensional structure of Sp100, which supports the mapping data and provides additional information on Sp100 structure/function relationships, was generated by computer modeling. Taken together, our studies indicate the existence of well defined Sp100 domains with functions in ND targeting, nuclear import, nuclear SUMOylation, and protein-protein interaction.

Amino Acid Sequence↗

PubMed2004

Subcellular localization of the ABCG2 transporter in normal and malignant human gallbladder epithelium.

Epithelium of the gallbladder and biliary tract is exposed to high concentrations of potentially harmful exogenous and endogenous compounds excreted into primary bile. As the ATP-dependent efflux pump ABCG2 can prevent cellular accumulation of anticancer drugs, estrogen sulfate, xenobiotics, porphyrins, and sterols, its expression in the biliary tract might mediate protection by hindering their penetration. We therefore investigated the expression and subcellular distribution of ABCG2 in normal and malignant human gallbladder. After demonstrating ABCG2 expression in gallbladder epithelium by RT-PCR and Western blotting, we analyzed the subcellular localization of ABCG2 by indirect immunofluorescence in gallbladder adenocarcinoma specimens, and compared it to that in cholelithiasis, and normal gallbladder samples (n = 54). In control, cholelithiasis, and well-differentiated tumor samples (grade 1, T1-3), ABCG2 is present at the luminal membrane of epithelial cells, which was proven by colocalization of apical-bound TRITC-labeled lectin (wheat germ agglutinin). In poorly differentiated gallbladder adenocarcinomas, intracellular ABCG2, in addition to luminal ABCG2 immunoreactivity, was found in 13/21 carcinoma samples (grade 2 and 3, T2-4, P < 0.01). In 3/11 of grade 3 tumors, ABCG2 was present in the cytoplasmatic compartment only (P < 0.01). In proliferating bile ducts of cholangiocarcinomas, ABCG2 showed an analogous staining pattern with presence in cytosolic compartments. However, the apical marker enzyme neutral endopeptidase remained on the membrane in all samples. To study whether phosphatidylinositol 3-kinase (PI3K) signaling might be necessary for ABCG2 membrane insertion, we treated freshly isolated human gallbladder epithelial cells with the PI3K inhibitor wortmannin. As assessed by indirect immunofluorescence, this maneuver redistributes ABCG2 to intracellular compartments. In conclusion, our data suggest a protective role for ABCG2 in well-differentiated gallbladder epithelial cells. Cytoplasmatic accumulation of ABCG2 in poorly differentiated carcinomas might coincide with malfunctioning of PI3K-signaling pathways during tumor progression.

ATP Binding Cassette Transporter, Subfamily G, Mem↗

PubMed2003

TOR regulates the subcellular localization of Ime1, a transcriptional activator of meiotic development in budding yeast.

The transcriptional activator Ime1 is a key regulator of meiosis and sporulation in budding yeast. Ime1 is controlled at different levels by nutrients and cell-type signals. Previously, we have proposed that G(1) cyclins would transmit nutritional signals to the Ime1 pathway by preventing the accumulation of Ime1 within the nucleus. We show here that nutritional signals regulate the subcellular localization of Ime1 through the TOR pathway. The inactivation of TOR with rapamycin promotes the nuclear accumulation and stabilization of Ime1, with consequent induction of early meiotic genes. On the contrary, the activation of TOR by glutamine induces the relocalization of Ime1 to the cytoplasm. Thus, TOR may sense optimal nitrogen- and carbon-limiting conditions to modulate Ime1 function. Besides TOR, ammonia induces an independent mechanism that prevents the accumulation of Ime1 in the nucleus. Both TOR and ammonia regulate Ime1 localization in the absence of Cdk1 activity and therefore use mechanisms different from those exerted by G(1) cyclins. Integration of independent mechanisms into a single early controlling step, such as the nuclear accumulation of Ime1, may help explain why yeast cells execute the meiotic program only when the appropriate internal and external conditions are met together.

Antibiotics, Antineoplastic↗

PubMed2001

Cellular and subcellular localization of Ras guanyl nucleotide-releasing protein in the rat hippocampus.

Ras guanyl nucleotide-releasing protein (RasGRP) is a recently discovered Ras guanyl nucleotide exchange factor that is expressed in selected regions of the rodent CNS, with high levels of expression in the hippocampus. Biochemical studies suggest that RasGRP can activate the Ras signal pathway in response to changes in diacylglycerol and possibly calcium. To investigate potential sites for RasGRP signaling, we have determined the cellular and subcellular localization of RasGRP protein in adult rat hippocampus, and have also examined the appearance of RasGRP mRNA and protein during hippocampal development. RasGRP immunoreactivity is predominately localized to those neurons participating in the direct cortico-hippocampo-cortical loop. In both hippocampal and entorhinal neurons, RasGRP protein appeared to be localized to both dendrites and somata, but not to axons. Electron microscopy of hippocampal pyramidal cells confirmed RasGRP immunoreactivity in neuronal cell bodies and dendrites, where it appeared to be associated with microtubules. The localization of RasGRP to dendrites suggests a role for this pathway in the regulation of dendritic function. Examination of developing hippocampal structures indicated that RasGRP mRNA and protein appear synchronously during the first 2 weeks of postnatal development as these neurons become fully mature. This result indicates that the RasGRP signal transduction pathway is not required during early hippocampal development, but is a feature of mature neurons during the later stages of development.

Aging↗

PubMed1995

Tissue distribution and subcellular localization of Na+ channel mRNA in the nervous system of the squid, Loligo opalescens.

Recent cloning of a putative Na+ channel alpha subunit cDNA, GFLN1, from the squid stellate ganglion has allowed us to study the expression of this ion channel at a cellular level. In situ hybridizations with a probe derived from and specific to 3' untranslated and coding sequence of GFLN1 were used to determine its tissue distribution as well as its subcellular localization. In sections of the stellate ganglion, the probe labeled all of the cells in the giant fiber lobe (GFL) and most cells in the cellular layer of the main ganglion. In these non-GFL portions of the stellate ganglion, labeling was particularly intense in the ventral large cells and weak or absent in the dorsal small cells. In the optic lobe, only a select group of cells, the second-order visual giant neurons, were intensely labeled. These results are consistent with electrophysiological data that show GFL-like Na+ currents in rare large cells dissociated from the optic lobe and in most but not all cells from the non-GFL part of the stellate ganglion. In sections of the subesophageal mass of the central nervous system, strong labeling for GFLN1 mRNA occurred in the fin lobe, posterior chromatophore lobe, central and latero-ventral palliovisceral lobes, and posterior pedal lobe. In all cases, labeling was detected only in the cellular layer of these tissues and never in nerves or neuropil. In situ hybridization with dissociated GFL neurons maintained in primary culture verified that Na+ channel mRNA is confined to the cell body. These results indicate that GFLN1 is expressed predominately in large cells with large or long axons, and that this mRNA is restricted to the cell bodies of these neurons.

Animals↗

PubMed2004

Expression of EGFP/SDCT1 fusion protein, subcellular localization signal analysis, tissue distribution and electrophysiological function study.

Full-length cDNA gene of sodium-dependent dicarboxylate co-transporter protein 1 (SDCT1) is cloned from normal human kidney tissue and inserted into EGFP (enhanced green fluorescent protein) expression vector along with N-terminal and C-terminal truncated SDCT1 genes, so to construct the eukaryotic expression vectors of EGFP/SDCT1 fusion proteins, which are transfected into human renal tubular epithelial cells (HKC). Subcellular localizations of these fusion proteins are observed by laser confocal microscope to determine the localization signal of the SDCT1 protein. Duplex PCR analysis validates that the fusion protein genes have been integrated into the genome of HKC. Western blot indicates that the fusion proteins have been expressed in HKC. Confocal microscopy analysis shows that human SDCT1 predominantly locates on the plasma membrane, which is consistent with the results predicted by bioinformatics approach; in HKC transfected with N-terminal truncated SDCT1 gene, the green fluorescence is mainly distributed on the plasma membrane; in HKC transfected with C-terminal truncated SDCT1 gene, the green fluorescence is mainly distributed in the cytoplasm. EGFP/SDCT1 mRNAs obtained by in vitro transcription are microinjected into Xenopus laevis oocytes for expression and the trans-membrane currents are measured by using two-microelectrode voltage-clamp technique. Na+ inward currents are present on cellular membrane of the injected oocytes. Immunohistochemical staining shows that human SDCT1 proteins are expressed on lumen membrane of the renal proximal tubule, but are negative in distal tubule, collecting duct, renal interstitium and glomerulus. The above-mentioned studies suggest that human SDCT1 protein is located on the lumen membrane of the renal proximal tubule, the C-terminal sequence of the SDCT1 is required for delivery and targeting localization, and the plasma membrane localization signal of the SDCT1 protein maybe locate in the C-terminal sequence.

Animals↗

PubMed2005

[Cloning and subcellular localization of apr-1--a new gene of tumor specific antigen family].

BACKGROUND & OBJECTIVE: apr-1 was cloned by improved polymerase chain reaction (PCR)-based subtractive hybridization from all-trans retinoic acid (ATRA)-induced apoptotic leukemia HL-60 cells in 1999. Preliminary results showed that apr-1 might be an apoptosis-related gene (GenBank ID: NM_014061). This study was to explore the background of apr-1 through gene cloning, bioinformatic analysis, and subcellular locating. METHODS: The cDNA encoding Apr-1 was amplified by reverse transcription-PCR (RT-PCR), and sequenced. Open reading frame (ORF) of apr-1 was analyzed with ORF finder software. Chromosome locus was defined by genome blast software. Conserved domains of amino acids were analyzed by protein blast software. Align (Cluster W) software in Vector NTI software package was used to analyze homogeneous genes (or proteins), and to draw the Phylogenetic Tree. Subcellular localization of apr-1 was performed. RESULTS: apr-1 was mapped to chromosome Xp11.22 with the ORF locating in 1 exon. Two MAGE conserved domains were found in Apr-1. Apr-1 shared homology with MAGE-A1, MAGE-B1, MAGE-C1, MAGE-D1, and Necdin. Phylogenetic analysis showed that Apr-1 was more closely related to MAGE-D1 and Necdin. Gene products of apr-1 were located in the nuclei of eukaryocytes. CONCLUSIONS: apr-1 is a member of MAGE family, and might belong to type II MAGE genes.

Amino Acid Sequence↗

PubMed2003

Subcellular localization of yeast ribonucleotide reductase regulated by the DNA replication and damage checkpoint pathways.

The fidelity of DNA replication and repair processes is critical for maintenance of genomic stability. Ribonucleotide reductase (RNR) catalyzes the rate-limiting step in dNTP production and thus plays an essential role in DNA synthesis. The level and activity of RNR are highly regulated by the cell cycle and DNA damage checkpoints, which maintain optimal dNTP pools required for genetic fidelity. RNRs are composed of a large subunit that binds the nucleoside diphosphate substrates and allosteric effectors and a small subunit that houses the di-iron tyrosyl radical cofactor essential for the reduction process. In Saccharomyces cerevisiae, there are two large subunits (Rnr1 and Rnr3) and two small subunits (Rnr2 and Rnr4). Here we report the subcellular localization of Rnr1-4 during normal cell growth and the redistribution of Rnr2 and Rnr4 in response to DNA damage and replicational stress. During the normal cell cycle, Rnr1 and Rnr3 are predominantly localized to the cytoplasm and Rnr2 and Rnr4 are predominantly present in the nucleus. Under genotoxic stress, Rnr2 and Rnr4 become redistributed to the cytoplasm in a checkpoint-dependent manner. Subcellular redistribution of Rnr2 and Rnr4 can occur in the absence of the transcriptional induction of the RNR genes after DNA damage and likely represents a posttranslational event. These results suggest a mechanism by which DNA damage checkpoint modulates RNR activity through the temporal and spatial regulation of its subunits.

DNA Damage↗

PubMed1999

Topology, subcellular localization, and sequence diversity of the Mlo family in plants.

Barley Mlo defines the founder of a novel class of plant integral membrane proteins. Lack of the wild type protein leads to broad spectrum disease resistance against the pathogenic powdery mildew fungus and deregulated leaf cell death. Scanning N-glycosylation mutagenesis and Mlo-Lep fusion proteins demonstrated that Mlo is membrane-anchored by 7 transmembrane (TM) helices such that the N terminus is located extracellularly and the C terminus intracellularly. Fractionation of leaf cells and immunoblotting localized the protein to the plant plasma membrane. A genome-wide search for Mlo sequence-related genes in Arabidopsis thaliana revealed approximately 35 family members, the only abundant gene family encoding 7 TM proteins in higher plants. The sequence variability of Mlo family members within a single species, their topology and subcellular localization are reminiscent of the most abundant class of metazoan 7 TM receptors, the G-protein-coupled receptors.

Amino Acids↗

PubMed1994

Subcellular localization and endocytic function of low density lipoprotein receptor-related protein in human glioblastoma cells.

The low density lipoprotein receptor-related protein (LRP) is a multifunctional cell surface receptor that binds and endocytoses several structurally and functionally distinct ligands. Several of the ligands for LRP participate in both normal physiology and pathophysiology of the central nervous system. To begin to gain insights into the role of LRP in the central nervous system, we have analyzed the expression, subcellular distribution, and endocytic function of LRP in human glioblastoma U87 cells. These cells express an abundance of LRP at both the mRNA and protein levels. A 39-kDa protein, which copurifies with LRP and regulates its ligand binding activity, is also highly expressed in U87 cells. The subcellular localization of LRP and the 39-kDa protein was analyzed using scanning laser confocal and electron microscopy combined with immunolabeled U87 cells. At the plasma membrane, LRP was largely confined to clathrin-coated pits. Within cells, LRP and the 39-kDa protein partially colocalized within rough endoplasmic reticulum and the Golgi complex, suggesting a potential intracellular interaction between the two proteins. Little 39-kDa protein was found in endosomes in which LRP occurred abundantly. In examining the functional role of LRP in U87 cells, we found that LRP at the cell surface and along the cellular processes was functional in the binding and endocytosis of its ligands, and its activity therein was regulated by the 39-kDa protein. Using truncated recombinant 39-kDa protein constructs, we also demonstrated that distinct regions of the 39-kDa protein were responsible for inhibiting the binding of different LRP ligands on U87 cells. Our results thus strongly suggest several potential roles for LRP in brain protein and lipoprotein metabolism, as well as control of extracellular protease activity.

Carrier Proteins↗